Therapy for hepatitis C virus (HCV) infection has advanced rapidly with the recent approval of several direct-acting antivirals. However, most of the DAAs in clinical use or clinical trials target the same stage of HCV replication cycle and are associated with rapid emergence of drug-resistant viral mutations. In addition, different HCV genotypes and clinical conditions may also require adjustment of treatment regimen. Therefore, there is still an ongoing need to develop new HCV inhibitors that target different stages of the HCV replication cycle, such as entry and assembly. Previously reported screening methods for HCV inhibitors either limit to a virus-specific function or screen chemical libraries at a single dose which usually leads to high false-positive or -negative rates. We developed a quantitative high-throughput screen (qHTS) assay platform with a cell-based HCV infection system. The highly sensitive assay can be miniaturized to 1536-well format for screening of large-scale chemical libraries. we performed a large-scale quantitative high-throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of about 350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial 3,000 molecule showing inhibitory activity for further structural analyses and functional assays. We were able to assign the majority of these compounds to specific stage(s) in the HCV replication cycle. These small molecules represent a diversity of chemotypes that are potential drug-like lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. Four compounds with novel structural and drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. We also screen a library of approved pharmaceutical collection of about 3,000 compounds (the NPC library). We identified chlorcyclizine HCl (CCZ), an over-the-counter drug for allergy symptoms, as a potent inhibitor of HCV infection. CCZ inhibited HCV infection in human hepatoma cells and primary human hepatocytes. The mode of action of CCZ is mediated by inhibiting the early stage of HCV infection, probably targeting viral entry into host cells. The in vitro antiviral effect of CCZ was synergistic with other anti-HCV drugs, including ribavirin, interferon-alpha;, telaprevir, boceprevir, sofosbuvir, daclatasvir, and cyclosporin A, without significant cytotoxicity, suggesting its potential in combination therapy of hepatitis C. In the mouse pharmacokinetics model, CCZ showed preferential liver distribution. In chimeric mice engrafted with primary human hepatocytes, CCZ significantly inhibited infection of HCV genotypes 1b and 2a, without evidence of emergence of drug resistance during 4 and 6 weeks of treatment, respectively. With its established clinical safety profile as an allergy medication, affordability and a simple chemical structure for optimization, CCZ represents a promising candidate for drug repurposing and further development as an effective and accessible agent for treatment of HCV infection. In an effort to optimize the CCZ class of compounds, we started a chemical/structural modification campaign, centered around chlorcyclazine, which resulted in optimized, non-chiral, nontoxic CCZ analogues with improved anti-HCV potency and pharmacokinetics that are able to provide good coverage in liver at very reasonable doses. Lead compounds inhibited HCVsc infection without affecting HCVpp entry or HCV replication in the replicon assay, which is similar to that of CCZ, suggesting unaltered mechanism of action. Lead compounds showing overall improved properties, will be selected for in vivo anti-HCV efficacy studies and potentially for further drug preclinical development efforts with the aim of moving additional compounds of this series toward anti-HCV human clinical trials. With the increasing pipeline of anti-HCV drugs in development, there is a need to establish an in vitro system to assess the efficacy of various combination therapies. To achieve this aim, we established an infectious full-length HCV system expressing a luciferase reporter (HCVcc-Luc) for combination testing of synergy, additivity or antagonism. We tested combinations between protease, NS5A, and nucleotide NS5B inhibitor classes in both Con1b replicon and HCVcc-Luc systems. Combinations between different classes show consistency across the two viral assay platforms and different computational softwares, MacSynergyII and CalcuSyn. Combinations between NS5A and nucleotide NS5B inhibitors were synergistic, while combinations of protease inhibitors with the other two classes were additive to antagonistic. Theoretically additive combinations were indeed additive in the HCVcc-Luc system. Subsequent application to combinations between these DAA classes and host-targeting agent cyclosporin A demonstrated additive to synergistic effects. Combinations between an entry inhibitor (S)-chlorcyclizine with these DAA classes or cyclosporin A were highlyly synergistic. Together, our results demonstrate applicability of this infectious HCV system in combination testing. This system allows for in vitro investigation of novel combinatorial regimens, including antivirals targeting the entry or assembly stage of the HCV life cycle. A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model.